title: Multiple mechanisms for E2F binding inhibition by phosphorylation of the retinoblastoma protein C-terminal domain.
abstract: The retinoblastoma protein C-terminal domain (RbC) is necessary for the tumor suppressor protein's activities in growth suppression and E2F transcription factor inhibition. Cyclin-dependent kinase phosphorylation of RbC contributes to Rb inactivation and weakens the Rb-E2F inhibitory complex. Here we demonstrate two mechanisms for how RbC phosphorylation inhibits E2F binding. We find that phosphorylation of S788 and S795 weakens the direct association between the N-terminal portion of RbC (RbC(N)) and the marked-box domains of E2F and its heterodimerization partner DP. Phosphorylation of these sites and S807/S811 also induces an intramolecular association between RbC and the pocket domain, which overlaps with the site of E2F transactivation domain binding. A reduction in E2F binding affinity occurs with S788/S795 phosphorylation that is additive with the effects of phosphorylation at other sites, and we propose a structural mechanism that explains this additivity. We find that different Rb phosphorylation events have distinct effects on activating E2F family members, which suggests a novel mechanism for how Rb may differentially regulate E2F activities.
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, CA 95064, USA.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Burke
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, CA 95064, USA.
authors.forename: Tyler J
authors.initials: TJ
authors.lastname: Liban
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, CA 95064, USA.
authors.forename: Tamara
authors.initials: T
authors.lastname: Restrepo
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, CA 95064, USA.
authors.forename: Hsiau-Wei
authors.initials: HW
authors.lastname: Lee
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, CA 95064, USA. Electronic address: srubin@ucsc.edu.
authors.forename: Seth M
authors.initials: SM
authors.lastname: Rubin
mesh_terms: D050684:E2F Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D016160:Retinoblastoma Protein / Q000378:metabolism*
chemical_list: D050684:E2F Transcription Factors; D016160:Retinoblastoma Protein
keywords: CDK; GST; HSQC; ITC; Rb protein; cell cycle regulation; cyclin-dependent kinase; cyclin-dependent kinases; glutathione S-transferase; heteronuclear single quantum coherence; isothermal titration calorimetry; multisite phosphorylation; protein–protein interactions